title: Decreasing lactate level and increasing antibody production in Chinese Hamster Ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases.
abstract: Large-scale fed-batch cell culture processes of CHO cells are the standard platform for the clinical and commercial production of monoclonal antibodies. Lactate is one of the major by-products of CHO fed-batch culture. In pH-controlled bioreactors, accumulation of high levels of lactate is accompanied by high osmolality due to the addition of base to control pH of the cell culture medium, potentially leading to lower cell growth and lower therapeutic protein production during manufacturing. Lactate dehydrogenase (LDH) is an enzyme that catalyzes the conversion of the substrate, pyruvate, into lactate and many factors including pyruvate concentration modulate LDH activity. Alternately, pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenases (PDHs), to be metabolized in the TCA cycle. PDH activity is inhibited when phosphorylated by pyruvate dehydrogenase kinases (PDHKs). In this study, we knocked down the gene expression of lactate dehydrogenase A (LDHa) and PDHKs to investigate the effect on lactate metabolism and protein production. We found that LDHa and PDHKs can be successfully downregulated simultaneously using a single targeting vector carrying small inhibitory RNAs (siRNA) for LDHa and PDHKs. Moreover, our fed-batch shake flask evaluation data using siRNA-mediated LDHa/PDHKs knockdown clones showed that downregulating LDHa and PDHKs in CHO cells expressing a therapeutic monoclonal antibody reduced lactate production, increased specific productivity and volumetric antibody production by approximately 90%, 75% and 68%, respectively, without appreciable impact on cell growth. Similar trends of lower lactate level and higher antibody productivity on average in siRNA clones were also observed from evaluations performed in bioreactors.
authors.affiliation: Department of Early Stage Cell Culture, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Meixia
authors.initials: M
authors.lastname: Zhou
authors.forename: Yongping
authors.initials: Y
authors.lastname: Crawford
authors.forename: Domingos
authors.initials: D
authors.lastname: Ng
authors.forename: Jack
authors.initials: J
authors.lastname: Tung
authors.forename: Abigail F J
authors.initials: AF
authors.lastname: Pynn
authors.forename: Angela
authors.initials: A
authors.lastname: Meier
authors.forename: Inn H
authors.initials: IH
authors.lastname: Yuk
authors.forename: Natarajan
authors.initials: N
authors.lastname: Vijayasankaran
authors.forename: Kimberly
authors.initials: K
authors.lastname: Leach
authors.forename: John
authors.initials: J
authors.lastname: Joly
authors.forename: Bradley
authors.initials: B
authors.lastname: Snedecor
authors.forename: Amy
authors.initials: A
authors.lastname: Shen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000917:Antibody Formation / Q000187:drug effects; D019149:Bioreactors; D016466:CHO Cells; D049109:Cell Proliferation / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D017186:Titrimetry
chemical_list: D003470:Culture Media; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D019344:Lactic Acid; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D017346:Protein Serine-Threonine Kinases; D005947:Glucose